The clinical utility of immunosuppressive drugs stems from their ability to prevent graft rejection following organ transplantation. Such drugs exert their effect by inhibiting T-cell activation, which apparently leads to a failure to activate the transcription of early genes that normally function in coordinating the various cells involved in the immune response (Schreiber et at., Immunology Today 13(4): 136-142 (1992)). Those immunosuppressive drugs referred to as immunophilin ligands, which bind specifically to the immunophilin class of endogenous intracellular receptors/binding proteins, differ considerably in structure, yet function similarly, acting as prodrugs, which, upon binding to an immunophilin, form an active complex that exerts a distal effect. For example, cyclosporin A and tacrolimus bind cyclophilin (Handschumacher et al., Science 226: 544-547 (1984); and Harding et al., J. Biol. Chem. 261: 8547-8555 (1986)) and FKBP (Harding et al., Nature 341: 758-760 (1989); Standaert et al., Nature 346: 671-674 (1990); Siekierka et al., Nature 341: 755-757 (1989); and Maki et al., PNAS USA 87: 5440-5443 (1990)), respectively, and the resulting active complex targets the protein phosphatase calcineurin (Liu et al., Cell 66: 807-815 (1991)), the binding of which inhibits T-cell activation. Rapamycin also binds FKBP, although the resulting active complex apparently targets a molecule other than calcineurin (Bierer et al., PNAS USA 87: 9231-9235 (1990)).
A major concern in immunosuppressive therapy associated with organ transplantation is the level of immunosuppressive drug circulating in the blood. Toxicity can result if the immunosuppressive drug level is too high; graft rejection and opportunistic infection can result if the immunosuppressive drug level is too low.
Current methods of assaying for immunosuppressive drugs in the blood involve the use of organic solvents, such as methanol and methylene chloride, to get rid of the immunophilins to which the immunosuppressive drugs bind. Such methods suffer from the disadvantages associated with organic extraction, e.g., performing the extraction procedure under a hood and disposing of the organic waste in accordance with environmental guidelines. In addition, the presence of an organic solvent in high concentration can denature an antibody used to assay the level of the immunophilin ligand in the blood. Although dilution of the organic solvent decreases the likelihood of antibody denaturation, it also dramatically reduces the sensitivity of the assay by so diluting the immunophilin ligand as to impair quantitation of the ligand.
Accordingly, there remains a need for a method of assaying for an immunophilin ligand in blood or blood components that is simpler to perform, i.e., fewer, less complex steps in less time, obviates the need for organic solvents, and provides increased sensitivity and precision over currently available assay methods. Therefore, it is an object of the present invention to provide such a method. Other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.